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Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study, published in peer-reviewed literature, minor restrictions in design and reporting, but otherwise adequate for assessment.

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Results and evaluations of 48 continuous breeding reproduction studies conducted in mice
Author:
Morrissey RE, Lamb IV, JC, Morris RW, Chapin RE, Gulati DK, Heindel JJ
Year:
1989
Bibliographic source:
Fundamental and Applied Toxicology, 13, 747-777
Reference Type:
study report
Title:
Unnamed
Year:
1985

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: NTP Reproductive Assessment by Continuous Breeding (RACB)
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): propylene glycol
- Substance type: organic
- Physical state: liquid
- Analytical purity: not specified
- Batch number: Lot # 9930 HK
- Supplier: Aldrich Chemical Company via Midwest Research Institute (MRI), Kansas City, MO
- Impurities (identity and concentrations): not specified, but it is stated that all impurities which equaled 1.6% or more were identified.
- Stability under test conditions: stable in the dosing vehicle (water) for up to 14 days

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, NY.
- Age at study initiation: 6 weeks
- Housing: group housed by sex in solid-bottom polypropylene or polycarbonate cages with stainless-steel wire lids, during the quarantine and the 1-week premating periods. subsequently, the animals were housed as breeding pairs or individually
- Diet (e.g. ad libitum): ground rodent chow, ad libitum
- Water (e.g. ad libitum): deionized/filtered water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Photoperiod (hrs dark / hrs light): 14/10

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Each concentration was mixed separately at periodic intervals.

VEHICLE
Water
- Concentration in vehicle: 1.00, 2.50 and 5.00%
Details on mating procedure:
In the continuous breeding study mice were exposed to the test substance for 7-day premating period, and were then randomly grouped as mating pairs and cohabited and treated continuously for 98 days. Data were collected on all newborns during this period within 12 hours of birth, after which each litter was discarded. After the 98-day cohabitation, the pairs were separated but continued on treatments. During the next 21 days, any final litters were delivered and kept for at least 21 days (weaning). The mother was dosed through weaning and F1 mice were dosed until mated at 74 ± 10 days of age. For this, male offspring were mated to female off-spring from the same treatment group (n = 20/group/sex) and the F2 litters were examined for litter size, sex and pup weight.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
14 days in the dose range-finding study; 7 days pre-mating period, 98 days (14 weeks) cohabitation, 21 days post-cohabitation. Any litters delivered during these 21 days (second generation animals) were kept for at least 21 days (weaning) and continued receiving the chemical treatment initially through lactation (mother was dosed throughout) and then through drinking water. Second generation animals were mated at 74 ± 10 days of age.
Frequency of treatment:
Daily
Details on study schedule:
Other: A 14-day dose-setting study utilized one control group and 5 groups of dosed animals (8/sex/dose). The endpoints for this study were clinical signs, mortality, body weight gain and consumption of food and water.
In the main study, the litters were removed and examined as soon as possible after delivery was completed. The offsprings were sexed, weighed and killed so that the female may be impregnated immediately. This approach maximized the number of litters which could be produced in the 14-week breeding phase.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 1.82, 4.80 and 10.10 g/kg bw/day (main study)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
0, 0.5, 1.0, 2.5, 5.0 and 10% in water (dose-range finding study)
Basis:
nominal in water
Remarks:
Doses / Concentrations:
5% in water (second generation animals)
Basis:
nominal in water
No. of animals per sex per dose:
Dose range-finding study: 8/sex/dose
Main study: 20/sex/dose in each treatment group; 40/sex/dose in the control group
Second generation animals: 20/sex/dose (only the highest dose (5% in water) was tested).
Control animals:
yes
Details on study design:
- Dose selection rationale: based on a 14-day dose-selecting study
- Rationale for animal assignment: stratified randomization procedure based on body weights.

Other: when significant adverse effects on fertility were observed in the continuous breeding phase a crossover mating trial was usually performed to determine whether F0 males or females were more sensitive to the effects. High-dose animals of each sex were mated to control mice of the opposite sex to determine the affected sex. The high dose animals were selected to increase the possibility of detecting effects in the crossover mating. There were three conbinations of control and treated mice: control males with control females, high-dose males with control females, and control males with high-dose females. The offspring of the crossover matings were analyzed as in Task 4, and the parents were necropsied. Results of mating high-dose mice with control group partners were compared to matings within the control group to determine which sex was adversely affected. The crossover mating was not done if significant reproductive effects were not observed in the continuous breeding phase.
Necropsies were performed in this series of studies, usually on only F0 mice involved in th crossover mating trial, when there was evidence of an effect on reproduction or, at the least, in the second generation even if there was no effects on the F0 mice. Endpoints examined for the females included selected organ weights and histology. At necropsy, the endpoints of male reproductive function included selected organ weights and histology, percentage motile sperm, epididymal sperm concentration, and percentage abnormal sperm. These multiple measured of fertility (whole animal, organ) were designed to increase the sensitivity of the RACB protocol.

Positive control:
Diethylstilbestrol and ethylene glycol monoethyl ether

Examinations

Parental animals: Observations and examinations:
Clinical signs, mortality, body weight gain and consumption of food and water were assessed during the 14-day dose-setting study.
Oestrous cyclicity (parental animals):
Not performed.
Sperm parameters (parental animals):
As no adverse effects on fertility were observed in the continuous breeding study, the subsequent substudy (crossover mating with subsequent examination of male reproductive function in F0 animals) was not performed.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
mean No. litters per pair, mean No. live pups per pair, mean No. live male pups per litter, mean No. live female pups per litter, proportion of pups born alive, sex of pups born alive, mean live pup weight per litter, mean live male pup weight per litter, mean live female pup weight per litter; adjusted mean live pup weight per litter; adjusted mean live male pup weight per litter; adjusted mean live female pup weight per litter, body weight.



Postmortem examinations (parental animals):
As no adverse effects on fertility were observed during the continious breeding study, subsequent crossover mating with subsequent necropsies of F0 animals was not performed.
Postmortem examinations (offspring):
Not performed.
Statistics:
Dose-related trend in fertility: the Cochran-Armitage test (Armitage, 1971)
Pairwise comparisons involving mating and fertility indices: Fisher's exact test
Overall differences in number of litters, number of live pups, proportion of live pups and the sex ratio for dose group means: Kruskal-Wallis test (Conover 1980)
Ordered differences in number of litters, number of live pups, proportion of live pups and the sex ratio for dose group means: Jonckheere's test (Jonckheere, 1954)
Pairwise comparisons of treatment group means: Wilcoxon-Mann-Whitney U test
Average pup weight: Kruskal-Wallis test
Treatment differences in average pup weight: analysis of covariance (Neter and Wasserman, 1974)
Pairwise comparisons: two-sided t test
Organ weights: analysis of covariance F test (overall equality) and t test (pairwise equality)
Reproductive indices:
Fertility index was determined as (No. fertile/No.cohabited) x 100
Mating index was determined as No. females with plugs/No. cohabited
Offspring viability indices:
No data.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No chemical-related deaths were observed in either a dose-range finding study or during the 14 weeks cohabitation period in the main study. No significant chemical related clinical signs of toxicity were noted during the main study cohabitation phase.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In the dose-range finding study, the untreated males, on the average, gained 5% of their original body weight. The correspoding value for animals in the 10.0% propylene glycol was 7%. The female CD-1 mice in the control and 10% dose groups gained 8 and 15% of their initial body weight, respectively. The comparison of average daily water consumption values revealed that animals in the 10.0% propylene glycol group consumed nearly 60% more than the control group.
In the main study, male mice in the control, 1.0, 2.5 and 5.0% dose groups gained an average of 8, 7, 10 and 7% of their original body weight after 14 weeks of treatment. Female mice responded similarly; group mean body weights for the females varied with the gestation phase. Dam weights at delivery were essentially the same for animals in the control and 3 treatment groups.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
In the dose-range finding study water consumption was greatly increased in the 10.0% group. It was suspected that treatment at 10.0% may result in significantly altered fluid balances and increased body weight realtive to the control group.
In the main study, the average daily consumption of dosed water by animals in the 3 treatment groups was consistently higher than the control values, especially in the highest dose group.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
For the second generation animals, estrual cyclicity was not sensitive to propylene glycol under the present experimental condition except a small decrease in the percent estrus phase in treated animals.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
For the second generation animals, sperm studies illustrated that propylene glycol treatment at the 5.0% dose level does not significantly affect (p > 0.05) sperm motility, sperm density (sperm count per g caudal tissue), or the incidence of abnormal sperm.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
All breeding pairs in the main study delivered at least 1 litter after 14 weeks of cohabitation. Propylene glycol administered in drinking water at up to the 5.0% level had no significant effect (p > 0.05) on any of the reproductive parameters. These included the number of litters per pair, the number of live pups per litter, the proportion of pups born alive, the sex ratio and both the absolute and adjusted live pup weights.
The cumulative number of days to litter were also uneffected by propylene glycol treatment.
For the second generation animals, the mating index (percent of plug positive/no. cohabited) for the control and treated animals was 85%. The fertility index (percent of no. fertile/no. cohabited) values for the control and treated pairs was 75 and 80%, respectively. No significant differences (p > 0.05) were noted with respect to litter size, proportion of pups born alive, sex ratio, as well as absolute and adjusted pup weights.

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks:
toxicity
Effect level:
10 100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No effects reported at the highest dose tested.

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Propylene glycol had no apparent effect on pup survival.

BODY WEIGHT (OFFSPRING)
Propylene glycol had no apparent effect on pup body weight gain. For the second generation animals treated with 5% solution of propylene glycol, there was no significant difference (p > 0.05) in the body weight between the treated and control animals.

ORGAN WEIGHTS (OFFSPRING)
For the second generation animals, the absolute as well as adjusted organ weights for the liver, kidney, seminal vesicles, right cauda, prostate, right testis and right epididymis were not affected by propylene glycol treatment.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
fertility
Generation:
F1
Effect level:
10 100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No effects on fertility of F1 generation were observed at the highest dose.

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOAEL
Remarks:
developmental effects
Generation:
F2
Effect level:
10 100 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: No effects on litter size, sex and pup weight in F2 pups were observed at the highest dose.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion